Surface plasmon resonance (SPR) microscopy is a technique that uses excitation of surface plasmons (SPs) to detect chemical and physical changes in a probed region adjacent to a sensing surface. A variety of sensors based on SPR techniques have been developed which provide a sensitive means of characterizing the thickness and index of refraction of ultrathin films occurring at the surface of a thin metal film. In recent years, SP sensors have been used extensively to characterize chemical and physical properties of a variety of biological materials and to probe binding events in real time. For example, SP sensors have been used successfully to characterize the morphology of a range of surfaces, probe the kinetics and dynamics of interactions between proteins, proteins and DNA and proteins and small molecules, monitor antibody-antigen binding and characterize DNA hybridization processes.
Surface plasmons, also know as surface plasmon waves or plasmon polaritons, are charge density waves, which propagate parallel to an interface between a conducting or semiconducting thin film and a dielectric sample layer. SPs are generated by coupling radiant energy from incident photons into the oscillating modes of free electrons present in a conducting material, such as a metal, or semiconductor material. SPs are highly localized at the surface of the conducting (or semiconducting) layer and the intensity of the electric field of a SP decays exponentially in directions perpendicular to the plane in which it propagates. The spatial distribution of a SP may be quantitatively described by a characteristic decay length corresponding to the distance over which the intensity of the SP decays to e−1 times its value at the conductor (or semiconductor)—dielectric sample layer interface. Decay length (L) is provided by the expression:                               L          =                      1                          2              ⁢                                                           ⁢                              Re                ⁡                                  (                                                                                    k                        sp                        2                                            +                                              k                        s                        2                                                                              )                                                                    ;                            (        I        )            wherein Re refers to the real part of the quantity in parentheses, ksp is the surface plasmon wavevector and ks is the wavevector in the dielectric sample layer adjacent to the conductor (or semiconductor). For a dielectric sample layer comprising water and a conducting thin film comprising gold the decay length is equal to about 83.1 nm for light having a wavelength of about 632.8 nm. The highly localized nature of SPs make them ideally suited for detecting very small changes in refractive index occurring in sensing regions proximate to a sensing surface (≦ about 300 nm).
In conventional SPR methods, a SP is excited by evanescent electromagnetic waves generated upon total internal reflection of an incident light beam. In the Kretschmann-Raether geometry, evanescent electromagnetic waves penetrate a thin metal film (≈50 nm) positioned between higher and lower refractive index dielectric layers and excite a SP, which propagates parallel to the outer surface of the metal film adjacent to the lower refractive index layer. The prism is needed to achieve the wavevector matching condition between the incident excitation light and the surface plasmons. For a given dielectric sample, photons of a certain wavelength and incident at a certain angle will generate evanescent waves that penetrate the metal layer and excite surface plasmons at the metal-dielectric sample interface. The intensity of reflected light will therefore be reduced and can be monitored as a signal of SP generation. Alternatively, in the Otto SPR configuration, the metal layer and prism are separated by an air gap and SPs are excited on the side of the metal film adjacent to the prism. A drawback of the Otto SPR configuration is that it is experimentally difficult to maintain a very thin and constant thickness air gap. Finally, in other SPR methods, surface plasmons are created by evanescent fields generated as light propagates down a fiber optic or waveguide having a thin metal interior layer
Excitation of SPs via total internal reflection is a resonant phenomenon that depends on the wavevector of the incident light (i.e. both the wavelength and angle of incidence of the incident light beam. In addition, excitation of SPs is dependent on the indices of refraction and thickness of the higher refractive index layer, lower refractive index sample layer and conducting (or semiconducting) thin film used to couple radiant energy into the oscillating modes of free electrons present in the conductor. The dispersion equation for a SP is provided by the equation:                                           k                          s              ⁢                                                           ⁢              p                                =                                    k              0                        ⁢                                                                                ɛ                    c                                    ⁢                                      ɛ                    d                                                                                        ɛ                    c                                    +                                      ɛ                    d                                                                                      ;                            (        II        )            wherein k0 is the free space wavevector (k0=ω/c); ∈c and ∈d are the complex permittivities of the conducting (or semiconducting) thin film and the lower refractive index dielectric sample layer, respectively and ω is the angular frequency. A resonance condition of exciting an SP is that the parallel component of the incident wavevector (kpar), must equal the surface plasmon wave vector (ksp):kpar=ksp  (III).The parallel component of the incident wavevector may be expressed in terms of the index of refraction of the medium in which the light is incident, n, the angle of incidence, θ, and the wavelength of the incident light beam, λ, by the equation for formation of a SP:                               k                      p            ⁢                                                   ⁢            a            ⁢                                                   ⁢            r                          =                                            2              ⁢                                                           ⁢              π              ⁢                                                           ⁢              n              ⁢                                                           ⁢                              sin                ⁡                                  (                  θ                  )                                                      λ                    .                                    (        IV        )            Substituting equations II and IV into equation III provides the following relationship expressing the resonance condition for the formation of a surface plasmon in terms of the angle of incidence and wavelength of the incident beam:                                           2            ⁢                                                   ⁢            π            ⁢                                                   ⁢            n            ⁢                                                   ⁢                          sin              ⁡                              (                θ                )                                              λ                =                              k            0                    ⁢                                                                                          ɛ                    c                                    ⁢                                      ɛ                    d                                                                                        ɛ                    c                                    +                                      ɛ                    d                                                                        .                                              (        V        )            As is evident from equation V, for a given metal film thickness and set of refractive indices of dielectric layers, the resonance condition may be satisfied by variation of either the angle of incidence or the wavelength of the incident light beam, or both.
In the derivation of the dispersion relation for the SP, equation II, two additional conditions that must be satisfied for surface plasmon generation to occur become apparent. First, SPs are p-polarized and so can only be excited by p-polarized incident light. And second, SPs are only supported at an interface made up of media with real permittivites of opposite sign.
As illustrated by equations II-V, changes in the refractive index of the dielectric sample layer adjacent to the thin metal film changes the resonance condition for generating a SP. This change in resonance condition may be monitored directly by measuring the intensity of the reflected incident beam as a function of angle of incidence, wavelength of the incident beam or both. Satisfaction of the resonance condition results in a sharp attenuation in the intensity of the reflected beam caused by a conversion of radiant energy of the incident beam into SPs at the interface between the thin metal film and the lower refractive index layer. Due to their spatially localized nature, SPs have also been used to excite photoluminescent materials. Specifically, energy from a SP is coupled to a photoluminescent material in a manner resulting in excitation of an electronic transition providing fluorescence or photoluminescence. An additional detector can be positioned in optical communication with the sensing surface to measure the intensity of fluorescence of materials pumped by the SPR process. The combination of attenuated reflectance SPR methods and SPR induced fluorescence has been demonstrated to provide a sensitive means of characterizing chemical and physical changes occurring at a senor surface.
Sensors based on SPR utilize the dependence of the SPR resonance condition on changes in the refractive index of a lower refractive index dielectric sample layer positioned adjacent to the thin metal (or semiconductor) film. In typical sensing applications, changes in the resonance condition for formation of SPs are monitored in real time and directly related to chemical or physical changes occurring at a sensing surface adjacent to the thin metal (or semiconductor) film. Sensors based on SPR may provide selective detection of materials and compounds by manipulating the chemical or physical properties of the sensing surface. In these applications, the sensing surface may be coated with a material exhibiting selective binding characteristics such that the refractive index varies in the presence of a specific material to be sensed. For example, the sensing surface may be made sensitive to a particular antibody by coating it with an antigen to that antibody. Using these principles, SPR detection has been successfully incorporated into a number of commercially available biological sensing devices including the sensors and screening devices manufactured by BIAcore, Inc.
Generally, a SPR optical configuration comprises (1) a source of electromagnetic radiation, (2) an optically transmissive component having a first refractive index, (3) a dielectric sample layer (or probe region) having a second refractive index less than that of the first refractive index of the optically transmissive component, (4) a conducting or semiconducting thin film positioned between the optically transmissive component and the dielectric sample layer (probe region) and (5) a detector. In this configuration, an incident beam is transmitted through the transparent region at an angle of incidence such that it undergoes total internal reflection at the interface between the optical transmissive component and the conducting thin film. The reflected incident beam is collected and directed to a detector capable of measuring its intensity as function of time. If the resonance condition outlined in Equations II to V is met, radiant energy is converted into a SP at the interface between the conducting or semiconducting thin film and the dielectric sample layer resulting in a measurable decrease in the intensity of the reflected incident beam.
Sensors based on SPR may utilize a number of different optical configurations. Exemplary optical configurations are described in Rothenhausler, B. and W. Knoll (1988). “Surface-plasmon microscopy.” Letters to Nature 332(14): 615-617., Hickel, W. and W. Knoll (1990). “Surface plasmon microscopy of lipid layers.” Thin Solid Films 187: 349-356, Hickel, W. and W. Knoll (1991). “Time and spatially resolved surface plasmon optical investigation of the photodesorption of Langmuir-Blodgett multilayer assemblies.” Thin Solid Films 199: 367-373, de Bruijn, H. E., R. P. H. Kooyman, et al. (1992), “Choice of metal and wavelength for surface-plasmon resonance sensors; some considerations.” Applied Optics 31(4): 440-442, de Bruijn, H. E., R. P. H. Kooyman, et al. (1993). “Surface plasmon resonance microscopy; improvement of the resolution by rotation of the object.” Applied Optics 32(13): 2426-2430, Berger, C. E. H., R. P. H. Kooyman, et al. (1994). “Resolution in surface plasmon microscopy.” Review of Scientific Instruments 65(9): 2829-2837 and Brockman, J. M., B. P. Nelson, et al. (2001) “Surface plasmon resonance imaging measurements of ultrathin organic films.” Annual Reviews of Physical Chemistry 51(1): 41-47, which are hereby incorporated by reference in their entireties to the extent not inconsistent with the present application.
The most common configuration in SPR sensing applications involves angle modulation of a substantially monochromatic, coherent incident light beam. In this technique, a surface plasmon resonance curve is generated by measuring the intensity of a reflected, substantially monochromatic, coherent incident beam, as the angle of incidence is systematically varied. Satisfaction of the SP resonance condition results in a measurable attenuation of the intensity of the incident beam corresponding to the minimum of a curve of reflected beam intensity versus incident angle. The angle corresponding to this minimum, referred to as the resonant angle (θsp), is dependent on the index of refraction near the surface of the conducting layer. Adsorption or binding of materials in the sensing region adjacent to the conducting layer changes the index of refraction in the sensing region and causes a measurable shift in the value of θsp. Quantification of the shift in θsp, therefore, provides a sensitive means of observing and characterizing changes in the composition and concentration of materials in sensing region. For example, studies have demonstrated linear correlations exist between resonance angle shifts and protein concentrations in the sensing region.
Despite the demonstrated effectiveness of angle modulation SPR techniques, theses optical configurations have several practical limitations. First, angle modulation optical configurations require use of complicated optical component rotation assemblies for selectably adjusting the angle of incidence of the incident beam. Typically, such assemblies provide for rotation of a combination of a light source, beam shaping optics and polarizing optics and/or rotation of a combination of light collection optics and a detector. Optical configurations requiring use of such complex rotation assemblies are undesirable because they are costly, spatially restrictive and require frequent maintenance and realignment. Second, use of complex optical component rotation assembles increases an instrument's sensitivity to optical misalignment caused by vibration and variations in ambient temperature and pressure. Finally, use of coherent light sources, such as lasers, in angle modulation SPR techniques results in unwanted optical interference of reflected beam components. Such optical interference is undesirable because it results in fringe patterns, which substantially degrades the optical quality of images obtained by SPR techniques.
Another optical configuration common to SPR sensing applications involves wavelength modulation. In wavelength modulation optical configurations, the intensity of the reflected incident beam is monitored for a fixed angle of incidence as the wavelength of the incident beam is systematically varied. In these techniques, a surface plasmon resonance curve is generated by measuring the intensity of a reflected incident beam, as the wavelength of the incident beam is varied. The wavelength corresponding to the minimum of a curve of reflected beam intensity verse wavelength, referred to as the resonant wavelength (λsp), indicates satisfaction of the resonance condition and is dependent on the index of refraction of a sensing region adjacent to the surface of the conducting layer. Quantification of the shift in λsp, therefore, provides a sensitive means of observing and characterizing changes in the composition and concentration of materials in sensing region. SPR wavelength modulation techniques commonly employ a constant angle of incidence and, therefore, do not require use of bulky optical rotation assembles.
Another application of SPR to sensing is SPR imaging techniques, wherein spatial differences in the reflectivity of an incident beam are measured as a function of time. In this technique, a collimated, monochromatic light beam is used for excitation of SPs and reflected light corresponding to a probe region is monitored by a two-dimensional array detector, such as a charge coupled device or camera. Differences in composition in the probe region are monitored in real time by observing a two-dimensional distribution of measured reflected light intensities. The thickness and refractive index of materials absorbed or bound to certain regions of the probe area may satisfy the SP resonance condition and provide for efficient SP formation. Therefore, these regions will exhibit attenuated reflected light intensities. Other regions of the probe area, in contrast, may comprise absorbed or bound materials having refractive indices which do not satisfy the SP resonance condition and do not result in efficient SP formation. Therefore, these regions will exhibit high reflectivities of the incident beam. Differences in the reflectivities of regions having different chemical and physical properties result in an image characterizing the entire probe area. The maximum contrast between regions in the probe area can be obtained by varying the imaging angle or wavelength of the SPR system.
Brockman, J. M., B. P. Nelson, et al. (2001). “Surface plasmon resonance imaging measurements of ultrathin organic films.” Annual Reviews of Physical Chemistry 51(1): 41-47 describes an optical configuration that is reported to improve quality and sensitivity of images generated by SPR imaging techniques. The authors disclose an optical arrangement comprising a collimated white light source, polarizer, prism—thin gold film sample assembly, narrow band interference filter and charge couple device (CCD) camera. The reference shows five SPR images corresponding to five different interference filters, which passes different wavelengths of excitation light. Although the authors report that SPR image quality may be optimized by selection of an interference filter having the appropriate transmission characteristics, the disclosed methods require time consuming, iterative image quality adjustment by manual removal and insertion of different interference filters. The authors principally depend on angle scanning to optimally contrast the samples in their probe region. Moreover, removal and insertion of optical interference filters requires repeated alignment of the excitation and detection optical arrangements. In addition, the teaching of the reference is limited to optical configurations providing discrete detection wavelength selection and does not provide the ability to tune the excitation or detection wavelength over a continuous range of values. Finally, the methods disclosed expose the sample to significant intensities of light having wavelengths not detected by the CCD camera, which do not contribute to SPR image formation and may damage materials in the probe region.
It will be appreciated from the foregoing that a clear need exists for methods and devices for generating SPs in thin conducting (or semiconducting films) which do not utilize angle modulation SPR, particularly angle modulation SPR optical configurations having complex rotational assemblies. Further, methods and devices for wavelength modulation SPR sensing and/or imaging having continuously tunable, incoherent light sources are needed. Finally, tunable SPR instruments are needed which eliminate undesirable optical interference problems and provide enhanced sensitivity and resolution.